During hepatic disease the liver has the unrivalled ability to regenerate, by activating mature hepatocytes which can divide and thereby reconstitute the functional liver mass. However in the context of chronic hepatocellular disease the liver can regenerate from an endogenous population of hepatic progenitor cells (HPCs). The mechanisms which are involved in the activation and differentiation of these HPCs is not fully understood. To investigate whether there is a differential signalling requirement in HPCs acquiring a biliary versus hepatocellular fate we established in the laboratory two models of chronic liver damage and regeneration, one of which causes hepatocellular death, and results in infiltrating HPCs regenerating hepatocytes, and a second which causes biliary blockage and death, resulting in biliary regeneration. Here we describe how during biliary regeneration the Notch signalling pathway is highly expressed and activated. HPCs cells are consistently associated with a myofibroblast niche which expressed the ligand Jagged-1 at high levels. We have modulated the Notch signalling pathway in both a co-culture system and our models in vivo to demonstrate that Notch signalling is important in the specification of biliary cells, and that inhibition of this pathway both in vitro and in vivo results in the abrogation of biliary commitment. During hepatocellular regeneration we have found that the negative repressor of Notch signalling Numb is highly expressed in tandem with a low expression of the Notch pathway. We suggest that Wnt signalling maintains Numb within these HPCs at a high level and that this, along with stimulation of a hepatocellular programme allows HPCs to exit from a biliary fate and assume a hepatocellular phenotype. Finally we have found that macrophage ingestion of debris promotes the expression of Wnt, and that ablation of these cells results in a phenotypic switch between HPCs assuming a hepatocellular fate and a biliary one.

No description available.

Les chondrocalcinoses articulaires (CCA) sont des arthropathies microcristallines caractérisées par la présence de cristaux de pyrophosphate de calcium dihydratés (PPCD) au sein du cartilage. Il existe trois formes de CCA : les formes familiales, transmises de façon autosomique dominante, et les formes sporadiques, dont la probabilité d'apparition augmente avec l'âge, et les formes secondaires à d'autres pathologies, comme l'hyperparathyroïdie. Le gène Ank code un transporteur exportant le pyrophosphate inorganique (PPi) hors des cellules. Un lien est clairement établi entre des mutations dans la séquence codante du gène Ank et les formes familiales de CCA Nous avons étudié la contribution du gène Ank dans la production de PPi par les chondrocytes articulaires stimulés avec du transforming growth factor-ß1 (TGF-ß1), leur sensibilité a ce facteur de croissance augmentant avec le l'âge des patients. Nous avons également, vu la présence fréquente des cristaux de PPCD chez les patients arthrosiques, et l'influence du niveau d'expression d'Ank dans la chondrogenèse, voulu élucider la possible implication d'Ank dans la perte du phénotype du chondrocyte articulaire, retrouvée dans l'arthrose. Enfin, nous avons étudié le rôle du calcium sur les effets du TGF-ß1 dans la production d'ePPi, l'hypercalcémie étant souvent retrouvée dans les tableaux cliniques d'hyperparathyroïdisme. Ces travaux ont permis de mettre en évidence le rôle majeur d'ANK en cas sensibilité accrue du chondrocyte au TGF-ß1. De plus, le PPi transporté par ANK influence le phénotype du chondrocyte articulaire. Enfin, le calcium joue un rôle crucial sur les effets inducteurs du TGF-ß1 dans la production de PPi. / Articular chondrocalcinosis (CCA) are microcrystalline arthtopathies characterized by the presence of calcium pyrophosphate dihydrate crystals (CPPD) in the cartilage. There are 3 forms of CCA, including the familal forms, dependent upon an autosomal dominant transmission mechanism, the sporadic forms, whose probability of occurrence increases with ageing, and the metabolic forms, secondary to other pathologies like hyperparathyroidism. The Ank gene encodes a transporter which exports inorganic pyrophosphate (PPi) outside the cells. A link is well established between mutations in the coding sequence of the Ank gene and the familial forms of CCA. We studied, on one hand, the contribution of the Ank gene in the production of PPi by articular chondrocytes stimulated with transforming growth factor-ß1 (TGF-ß1), since the sensitivity of chondrocytes to this growth factor increases with ageing. On the other hand, since CPPD are often associated with osteoarthritis (OA), and since Ank expression levels influences the chondrogenesis, we elucidated the possible implication of Ank in the loss of the articular chondrocytes phenotype observed during OA. Finally, we analyzed the role of calcium on the TGF-ß1 effects on ePPi production, hypercalcaemia being often found during hyperparathyroidism. This work allowed demonstrating, on one hand, the predominant role of ANK in the situations of chondrocytic hypersensitivity to TGF-ß1. On the other hand, the PPi exported by ANK influences the expression of articular chondrocytic markers. Finally, calcium plays a crucial role in the inducer effects of TGF-ß1 in the production of PPi.

ABSTRACT MUC1, a mucin-like transmembrane glycoprotein, is highly overexpressed and aberrantly localized in several invasive carcinomas. MUC1 is proposed to play numerous roles in the transformed behaviour of cells in which it is expressed. A number of these roles are facilitated by the interaction of MUC1 with β-catenin, a protein that is central to both cellular adhesion as well as Wnt-responsive gene transcription. The aim of this study was to investigate MUC1 expression, localization, and interaction with β-catenin, as a means of providing insight into the behaviour of human oesophageal squamous cell carcinoma. This cancer-type is exceptionally aggressive and is a major cause of cancer-related morbidity and mortality in South Africa. MUC1 is expressed and aberrantly localized in oesophageal squamous cell carcinoma cell lines, as demonstrated by RT-PCR, western blotting and indirect immunofluorescence. Moreover, evidence from coimmunoprecipitation assays shows that the MUC1 cytoplasmic tail and β-catenin form a complex both at the cell membrane and importantly, within the nucleus of these cell lines. This is the first demonstration of such a complex in the nucleus of a carcinoma derived from stratified, as opposed to simple, epithelia. Data presented here further indicates that activation of the epidermal growth factor receptor results in modulation of the association between MUC1 and β-catenin at the cell membrane. MUC1 membrane-localization, and interaction with β-catenin, may modulate cellular adhesion through steric interference of cell surface adhesion molecules as well as through sequestration of β-catenin away from adherens junctions. On the other hand, MUC1 association with β-catenin may enhance β- catenin signalling either through the stabilization of β-catenin, or as an essential functional component of the β-catenin/LEF/TCF transcription factor complex. Furthermore, results presented in this study identify oesophageal squamous cell carcinoma as a prime candidate for MUC1-specific immunotherapy. This finding is of substantial importance considering the ineffectual nature of existing therapies used in the treatment of oesophageal carcinoma.

1 / Rongrong Zhang

The process of vertebrate development requires communication among many cells of the embryo in order to define the body axis (front/back, head/tail or left/right). The Wnt signaling network plays a key role in a vast array of cellular processes including body axis patterning and cell polarity. One arm of the Wnt signaling network, the non-canonical Wnt pathway, mediates intracellular Ca2+ release via activation of heterotrimeric G proteins. Regulator of G protein Signaling (RGS) proteins can accelerate inactivation of G proteins by acting as G protein GAPs and are uniquely situated to control the amplitude of a Wnt signal. I hypothesize that individual RGS proteins are critical in modulating the frequency and amplitude of Wnt/Ca2+ signaling in different tissues and at different developmental stages and this modulation is essential for developmental patterning events. To this end, this thesis is focused on the effects G protein regulation has on intracellular Ca2+ release dynamics and developmental patterning events. I combine cellular, molecular and genetic analyses with high resolution Ca2+ imaging to provide new understanding of the role of RGS proteins on Wnt mediated Ca2+ release dynamics and developmental patterning events. In chapter 2, I describe endogenous Ca2+ dynamics from the very first cell divisions through early somitogenesis in zebrafish embryos. I find that each phase of zebrafish development has a distinct pattern of Ca2+ release, highlighting the complexity of Ca2+ ion and cellular physiology. In Chapter 3, I identify rgs3 as potential modulator of Ca2+ dynamics and Chapter 4 expands upon these observations by providing data supporting that Rgs3 function is necessary for appropriate frequency and amplitude of Ca2+ release during somitogenesis and that Rgs3 functions downstream of Wnt5 activity. My results provide new evidence that a member of the RGS protein family is essential for modulating the non-canonical Wnt network to assure normal tissue patterning during development. In Chapter 5, I report the identification and characterization of Rgs3b, a paralogue to Rgs3, in zebrafish. I describe results indicating that Rgs3b is poised to interact with Wnt11 indicating that individual RGS genes may have unique roles in modulating Wnt/Ca2+ signaling in different tissues or different stages. In conclusion, this thesis provides data supporting that individual RGS proteins are critical in modulating the frequency and amplitude of Wnt/Ca2+ signaling in different tissues and at different developmental stages and this is a substantial breakthrough in understanding how RGS proteins function to fine-tune known signaling pathways

Breast and pancreatic cancers are among the major causes of cancer mortality in our society. There has been a significant decline in mortality from breast cancer over the last two decades, while pancreatic cancer has an exceptionally poor prognosis. Although these malignancies have very different clinical outcomes they share the common feature that metastatic disease is almost uniformly fatal. The existence of cancer stem cells has been postulated as a major factor in tumour recurrence after traditional chemo- or radio-therapy. Addressing this important clinical question requires a deeper understanding of the biology of normal and cancer stem cells and the signalling pathways involved in their regulation. The identity of the pancreatic stem cell remains elusive. However, using a murine model of haematopoietic stem cell (HSC) transplantation I have demonstrated for the first time transdifferentiation of these bone marrow derived cells into mature pancreatic acinar cells, where they appear to contribute to cell turnover ultimately forming acini and lobules. These data show that HSC have surprising developmental plasticity and provide insight into a potential stem cell niche in the pancreas. The Hedgehog, Wnt and Notch signalling pathways play a critical role in early development and in the maintenance and self-renewal of stem cells. There is also increasing evidence that dysregulation of these pathways contributes to the development of many malignancies. There is relatively little information regarding their role in breast cancer development and progression. I used immunohistochemistry for key proteins in these pathways, sonic hedgehog, beta-catenin and Notch 1 in three substantial series of human breast lesions and determined that abnormal expression of these proteins is an early event in the development in breast cancer, and is associated with particular breast cancer subtypes, Shh and beta-catenin expression is associated predominantly with the basal-like phenotype and Notch 1 with the HER2 amplified phenotype. Overexpression of Shh in particular confers a worse clinical outcome in invasive ductal carcinoma. Furthermore, increased levels of Shh in a 3D culture model of non-transformed mammary epithelial cells resulted in disorganisation of acini and the development of an abnormal discohesive phenotype. Finally the role of Shh was investigated in a mammary epithelial transplantation model, where overexpression of Shh resulted in the development of hyperplasia of the mammary ductal epithelium. Together these data confirm that the Hedgehog, Wnt and Notch developmental pathways are dysregulated in breast cancer and represent viable targets for further investigation of potential novel therapies in breast cancer.

Aberrant activation of the canonical Wnt signaling pathway accounts for the vast majority of colorectal cancers. The Rac1 GTPase is overexpressed in colon cancer, and its splice variant, Rac1b, is preferentially expressed in colon tumours. Rac1 and Rac1b have both been previously shown to crosstalk with the canonical Wnt signaling pathway in colon cancer; however, the specific means by which this crosstalk occurs were unclear. This study examines the molecular mechanisms of Rac1/1b in the cooperation with canonical Wnt signaling in colon cancer. In a colon cancer cell line with dysregulated Wnt signaling, the constitutively active Rac1 mutant, V12Rac1, was observed to transcriptionally upregulate the expression of a gene set associated with cellular migration. Further, V12Rac1-mediated promotion of cell migration was dependent on its nuclear localization. Previous work in our lab has shown a Rac1-specific activator, Tiam1, is present in the nucleus at the promoter of Wnt target genes upon Wnt3a stimulation; and that exogenous introduction of Tiam1 increased the expression of a Wnt-responsive reporter (TopFlash). Given the importance of nuclear localization of Rac1 in the promotion of tumourigenic processes, we demonstrated that knockdown of endogenous Tiam1 reduced TopFlash expression, proving reverse specificity and strengthening the evidence of a nuclear role for Rac1. Since some functional differences exist between Rac1 and Rac1b, we also examined Rac1b for transcriptional targets following induction, and identified the RhoA effector, ROCK2, which has been previously associated with cell migration. ROCK2 demonstrated a positive correlation with Rac1b transcript expression in primary colon tumours as compared to matched normal tissue specimens. Interestingly, the observed induction in ROCK2 transcript did not translate into a detectable change in protein expression or kinase activity. Like Rac1, Rac1b also promotes cellular motility, which is dependent on nuclear localization. Cell migration can be negatively regulated by E-cadherin. Following Rac1b knockdown in HT29 cells, we show that Rac1b might contribute to motility through upregulation of the E-cadherin-repressor, Slug. Taken together, we provide greater insight into the mechanistic roles of Rac1 and Rac1b in transcriptionally regulating target genes to promote cellular processes, such as cell migration, in colon cancer with dysregulated canonical Wnt signaling.

Wnt signaling is important in human development and disease, thus dysregulated beta-catenin constitutes an attractive target for drug intervention. The few functional inhibitors currently available target transcriptional activation, therefore, identifying novel upstream modulators would be of tremendous importance to elucidating the mechanisms involved in regulatingbeta-catenin activity. To achieve this, I developed a high-throughput screen to assess beta-catenin stability in mammalian cells using a luciferase tagged beta-catenin molecule. This assay was used to screen three chemical libraries to identify small molecule modulators of the pathway. Identified inhibitors/activators of the pathway were investigated via secondary assays. The most promising inhibitor, 21H7, significantly attenuated activated beta-catenin signaling in colon cancer cells, decreasing beta-catenin stability. The inhibitory effects of 21H7 and a structurally similar compound were shown to not only inhibit Wnt target gene expression in colon cancer cells, but also prostate cancer lines. Thus, 21H7 represents an attractive lead compound for further study.

Wnt signaling is important in human development and disease, thus dysregulated beta-catenin constitutes an attractive target for drug intervention. The few functional inhibitors currently available target transcriptional activation, therefore, identifying novel upstream modulators would be of tremendous importance to elucidating the mechanisms involved in regulatingbeta-catenin activity. To achieve this, I developed a high-throughput screen to assess beta-catenin stability in mammalian cells using a luciferase tagged beta-catenin molecule. This assay was used to screen three chemical libraries to identify small molecule modulators of the pathway. Identified inhibitors/activators of the pathway were investigated via secondary assays. The most promising inhibitor, 21H7, significantly attenuated activated beta-catenin signaling in colon cancer cells, decreasing beta-catenin stability. The inhibitory effects of 21H7 and a structurally similar compound were shown to not only inhibit Wnt target gene expression in colon cancer cells, but also prostate cancer lines. Thus, 21H7 represents an attractive lead compound for further study.